Germicide



, 2,198,899 PATENT OFFICE GERMICIDE 1 William Nelson Axe and Douglas D. Henson,

Austin, Tex,

assignors to Union Oil Company of California, Los Angeles, Calif., a corporation of California No Drawing. Application July 20, 1937, Serial No. 154,626

4 Claims.

This invention relates to a germicide or bactericide and the method of producing the same. More specifically the invention relates to a germicide or bactericide from nitrogen bases recovered from certain petroleum fractions.

It has been discovered that nitrogen bases recovered from certain petroleum fractions possess germicidal properties which render them especially valuable as bactericides. These nitrogen bases exist in many crude oils and are found in appreciable quantities in asphalt base oil, such as California petroleum.

The invention, therefore, resides in recovering the nitrogen bases from petroleum and producing a bactericide from said bases, In producing the bactericide or germicide the bases are converted to a water soluble form by conversion to a readily water soluble salt. Treatment of the bases with a mineral acid, such as dilute hydrochloric acid produces a salt which is highly water soluble. Other mineralacids, such as hydrobromic, hydroiodic, sulphurous, phosphoric, or sulphuric acid may be employed to convert these bases into a water soluble form. However, I prefer to use dilute hydrochloric acid. Q'I'hese bases dissolved in dilute hydrochloric acid exhibit appreciable bactericidal action. However, I find that if these bases are .precipitated from solution in a high state of dispersion then bactericidal efficiency is materially increased.

v It is, therefore, an object of the present invention to recover nitrogen bases from petroleum,

' or other allied sources, such as coal tar, and

then produce a germicide or bactericide from said bases by forming a dispersion of said bases. I

It is another object of this invention to convert' nitrogen bases into water soluble salts, dissolve said salts in water and then form a germicide from said solution by precipitating the bases from said solution in a fine state of dispersion.

It is another object of the invention to produce a germicide by converting nitrogen bases into a water soluble form by treatment with a mineral acid, such as dilute hydrochloric acid, dissolving the acid treated bases in water and precipitating the bases therefrom by the addition of a buffer agent. As bufier agents I may use soluble peptones or other natural or prepared bufier agents. Sodium acetate-hydrochloric acid buffers of pH between 3.0 to 4.5 will precipitate the bases. Since peptone and horse serum are capable of bringing about precipitation it is believed that any soluble protein will accomplish the same result. Natural buffers are preferred in the preparation of germicides because of their non-toxic nature. The most available protein for this purpose is gelatin.

Other objects will appear from the following description:

Bases from petroleum distillates are of unusual pharmacological interest because, unlike volatile bases from other natural sources, they contain in decidedly preponderant amount non-aromatic types of unknown structure. For many of these products a cyclopentane, or so-called naphthenic structure, is highly probable. nets of plant occurrence, they would be classed as alkaloids; however, they differ from the most important plant alkaloids in being volatile and oxygen free.

During the exhaustive investigation of petroleum bases their apparent low toxicity has sug-- gested the possible discovery of products of therapeutic value; so, to determine the particular fractions and types of these bases of greatest promise for a pharmacological study, a determination of their germicidal action appeared desirable. Since experiment has indicated that the higher boiling bases containg the most potent bactericidal material, a study of transformer: oil bases was inaugurated. After a preliminary examination of the bactericidal action of various base fractions, the following determinations were made on a promising aromatic and likewise on a non-aromatic portion having approximate average boiling points of 340 C.: (1) accurate phenol coefiicients at 20 and 37 C. against strains of Eberthella typhi and Staphy ococcus aureaus; (2) the effect of horse serum on their germicidal action; (3) mycostatic action against the above mentioned bacterial; (4) the effect of a small amount of the bases on agar plates heavily seeded with bacterial; (5) the action of the total acidity of the hydrochloride solutions on bacteria.

In refining California asphalt base petroleum transformer-oil stock with liquid sulphur dioxide, this solvent withdraws the small amount of contained nitrogen bases and, after its recovery, the residual volume of hydrocarbon oil constitutes what is known as an Edeleanu extract. Five hundred barrels of this extract was processed as follows: The bases were extracted with 15% sulphuric acid, then liberated with sodium hydroxide and finally dividedv into three fractions (I, II, III) by vacuum distillation from a laboratory shell still. The yield of bases was 3% barrels.

TABLE I.--Physical constants of transformer-oil bases Fraction 1 N 25 1) D 15. 5 4

Stock laboratory cultures of E. typhi (Rawlings strain) and Staph. aureus (a laboratory strain) were used in all tests. Although the Rawlings strain is not the standard recommended in the Were these prod- F. D. A. method for the determination of phenol coeflicients, the remarkable constancy, observed by other investigators and verified in this work, justifies its use. The resistance of the E. typhi culture employed fell within the prescribed limits in every instance over a period of six months. The minimal resistance usually prescribed for Staph. aureus is survival of the bacterial for five minutes in a 1:60 dilution of phenol, whereas the particular culture used in these tests showed a slightly weaker but constant resistance. Growth after contact for five minutes in a 1:70 dilution of phenol was observed in each control run. At 37 C. a resistance similar to the conditions stated above appeared with phenol dilutions of 1:100, whereas the F. D. A standard resistance limit should be found with a dilution of 1:80.

Since transformer-oil bases are insoluble in water, a readily soluble salt was selected for this work. Solutions of these bases in dilute hydrochloric acid exhibited considerable bactericidal efficiency. However, under the experimental conditions employed the peptone 1n the culture medium, by virtue of its bufier action, precipitates the petroleum bases in a high state of dispersion; accordingly, it should be emphasized that our measurements apply to free bases liberated in situ as a fine emulsion,

The stock solutions of the bases were made up with standard N/ 10 acid, h nceltheacidity of any particular dilution was known quite accurately.

Although the wide variation in phenol coeflicients of a series of fractions having the same acid content apparently precluded the possibility of the acid being the active germicidal agent, this conclusion was confirmed by control tests with the bases omitted.

Fraction I (Table I) in 6 N hydrochloric acid was extracted with chloroform. In this way, the aromatic hydrochlorides were segregated in the aqueous layer and the non-aromatic hydrochlo rides in the chloroform layer. The liberated aromatic bases (6.5% of the total) had an N 25/D value of 1.6020- and for the non-aromatics this value was 1.5364.

The aromatic bases were separated into 27 fractions by distillation through a reflux column under diminished pressure. Stock solutions of (l) the crude bases, (2) the unfractionated nonaromatics and (3) aromatic fractions 2 and 2'7, were prepared by dissolving 1 g. samples in just the requisite amount of N/lO hydrochloric acid. The results obtained from these preparations are compiled in Table II.

TABLE H.Phenol coemcients of transformer-oil In order to determine if any exaltation in germicidal power occurs in the higher boiling bases, the original fractions II and III (Table I) were distilled under reduced pressure from a Claisen flask, the distillate from each sample being collected in two equal portions. Stock hydrochloride solutions were prepared as before and used in arriving at the following phenol coefficients:

TABLE Ill-Phenol coefllcients of mired type bases P1181101 00- Fraction efliclent (F. D. A)

III 6. 4 II: 8.0 III; 9. 0 In. 10.6

TABLE IV.-.Distillation data of aromatic transformer-oil bases Distilling Fraction temp. at N 25/D Since dilutions of these fractions showed no exaltation in germicidal power over the more well defined aromatic cuts of Fraction I, their exact coefiicients were not determined.

From 100 cc. of regained non-aromatic bases (III) distilled under 23 mm. pressure, cc. was collected in 10 cc. portions and the phenol coeflicients of fractions 1, 4, 5, 6 and 8 in dilute hydrochloric acid were determined.

TABLE V.-Dz'stil loti0n data and phenol coefiicients of non-aromatic transformer-oil bases Distilling Phenol co- Fraction temp. at N 25/1) efiicient 23 mm. (F. D. A)

The phenol coeflicients in Table V suggest that certain individual non-aromatic components in the higher boiling ranges possess exceptional germicidal properties. However, it is Worthy of note that fraction 5 has the maximum coeflicient while the first and last cuts show identical values.

Phenol coeflicients of fractions L121 (Table II) and IIINAs (Table V) Stock 1:100 dilutions were made as follows: one gram of bases was dissolved by the drop-wise addition of standard N/10 hydrochloric acid. The acid required for IAZT and HINAS solutions was 62.46 cc. and 65.82 00., respectively. After removal of tarry matter and hydrocarbon oil, the solutions were diluted to cc. with sterile distilled water. Although this procedure does not reveal the exact amount of bases in solution, reproducible results were possible. Furthermore, since all of the subsequent data are based on the original one gram of material, the inherent error favors the resistant action of the bacteria rather than the germicidal effect of the bases. pH values of the stock solutions, as estimated colorimetrically, were 3.8 and 4 for Lm and IIIms, respectively.

Phenol coefl'lcients of the two fractions under consideration were redetermined with care using E. typhi as the test organism at 20 C., and were found to check the values previously listed. In these tests dilutions were prepared in volumetric flasks rather than in the medication tubes.

TABLE VII.--Phenol coeificients of I427 and IIIms "Growth observed in h tubes containing 1: 25 dilution of the base.

The mycostatic tests described herein were carried out according to standard procedure. Preliminary tests were made, using the free In and IIINA5 emulsified in agar medium. The tubes, cooled in slants, were streaked with a 4 mm. loop holding a broth culture. Under these conditions growth of E. typlhi and Staph. aureus was not inhibited.

The inhibiting action of base emulsions formed in situ from the hydrochloride solutions was also studied. One cubic centimeter of various dilutions was added to sterile test-tubes with subsequent addition of 5 cc. of beef extract agar. The content of the tubes was thoroughly mixed and then allowed to harden in slants. Phenol dilutions .were made up in an. analogous manner, thus affording a basis of comparison. Blank tubes containing only agar medium served as. controls. The slants were streaked with a 4 mm. loop as uniformly as possible. The results in Table VIII refer to a 48 hour incubation per-0d.

\TABLE VIII.Inhiln'tion tests using E. typhi' and Staph. aureus Stock Final dilution dilution E. typM Staph. aumu 1-100- 1-600 Complete inhibition. Complete inhibition. 1-300- 1-800 Slight growth. Slight growth. 1-400- 1-2400 Normal growth Do. 1-500 1-3000 -do Do.

IIINM 1-100- 1-600 Completeinhibition Complete inhibition. 1-1200. 1-7200 Normal growth. Do. 1-1300.- 1-7800 do Do. 14400.... Do. 1-1500. 1-9000 Slight growth. 1-1800- Do. 1-2000.--. Do.

Phenol 1-60 Slight growth. 1-70 Do. 1-80 Do. 1-90 Normal growth. 1-100- 1-600 o D0. 1-110- 1-660 Normal growth- .I Do.

The I Tests on heavily seeded agar plates Here the usual beef extract peptone agar was employed. Each plate contained 0.2 cc. of a 24 hour broth culture and 15 cc. of the agar medium. ".lhev base dilutions were applied by means of a single streak of a 2 mm. loop and the readings taken after a 48 hour incubation period. The plate containing E. typhi streaked with Lm showed only a faint clear zone and the Staph. aureus plate streaked with the same dilution remained unchanged; in contrast, the 1:100 dilution of IIIims gave well defined clear zones in both the E. typhi and Staph. aureus plates. Phenol dilutions of 1:90, 1:100 and 1:1000 were applied in like manner without any observable effect on the bacterial growth. In an additional test with Staph. aureus the germicides were applied to the heavily seeded plates through a saturated circular filter paper 6 mm. in diameter. Again IA27 was without effect in dilution of 1:100. However, IIIms showed a clear zone in 1:100 and a faint clear zone at 1: 1400 dilution.

Nitrogen bases, compare very favorably with phenol as a bactericide. A resume of the inhibition experiments brings out the fact that growth of E. typhi of standard virulence is completely inhibited by sample Lin and sample IIINAS in dilutions of 1:600, as compared with 1:480 for *phenol. Although sample Lm was inefiective Staph. aureus in the usual germicide tests, it inhibits the growth of the organism in dilution of 1:600. Likewise, mNAS is highly efficient in this respect, inhibiting completely the growth of Staph. aureus in a dilution of 118400.

Experiments with ordinary emulsions of the free bases showed no appreciable germicidal action. Also it may be pointed outthat emulsions of free bases in agar medium possess no inhibiting action against the bacteria used. However, in all of the phenol coefficient determinations a very pronounced turbidity, due to liberation of free bases, takes place upon addition of the broth culture. The buffer action of the peptone evidently takes up the hydrochloric acid, liberating the bases in a finely dispersed state. With respect to the inhibition tests, a similar action occurs with the broth-agar medium. The molten agar becomes perfectly opaque due to the liberated bases, yet inhibitory action is quite pronounced. Thus it would seem that an extremely fine emulsion is germicidal; whereas the macro droplets produced by agitating the bases with soap solution are without effect.

We claim:

1. In a process for the production of cide from water-soluble nitrogen base salt solutions obtained by treating nitrogen bases with a mineral acid and dissolving the soluble salts thus produced in water, the step of adding a buffer agent to said aqueous solution to precipitate the bases from solution in a fine state of dispersion.

2. A process according to claim 1 in which the nitrogen base is precipitated from the aqueous solution by means of a buffer agent having a pH range between 3.0 and 4.5.

3. A process according to claim 1 in which a soluble peptone is added to the aqueous solution of the nitrogen base salt to precipitate the nitrogen base therefrom.

4. A process according to claim 1 in which gelatin is added to the aqueous solution of the nitrogen base salt to precipitate the nitrogen base therefrom.

WELIAM NELSON AXE. DOUGLAS D. HENSON.

a germi- 

